Generation of Dendritic Cells In Vitro From Peripheral Blood Mononuclear Cells With Granulocyte-Macrophage-Colony-Stimulating Factor, Interleukin-4, and Tumor Necrosis Factor-α for Use in Cancer Immunotherapy

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Abstract
The purpose of the study was to characterize the requirements in terms of precursors, developmental pathways, and media for the generation of large numbers of mature dendritic cells (DC) under conditions acceptable for use in adjuvant, active immunotherapy strategies for surgically treated malignancies. Although limited previously by the small numbers accessible, DC-based immunotherapies for malignancy have become more realistic with the development of methods for efficiently generating larger numbers of DC from peripheral blood mononuclear cells (PBMC) in vitro, but these methods rely on clinically unacceptable culture conditions (such as inclusion of fetal bovine serum), necessitating the development of methods for generating functionally equivalent DC in serum-free conditions. Plastic-adherent PBMC (from healthy donors and patients with cancer) were incubated for 7 days with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) with and without tumor necrosis factor-α (TNF-α) in fetal bovine serum-containing and serum-free media and were analyzed by Wright's stain for morphology, flow cytometry for phenotype, and mixed lymphocyte reaction for allostimulatory function. Growth in either serum-containing or serum-free media supplemented with GM-CSF and IL-4 yielded a similarly heterogeneous population of cells, 6% to 10% of which had the morphology (large cells with thin projections), immunophenotype (including CD83+), and function of mature DC. Tumor necrosis factor-α significantly augmented the number of these mature DC, whereas preculture depletion of CD14+ PBMC virtually eliminated them. Generation of mature DC in the authors' serum-free clinically applicable conditions is similar to serum-containing conditions and requires CD14+ precursors, differentiation through a CD14-CD83− immature stage under the influence of GM-CSF and IL-4, and maturation into a CD83+ DC under the influence of TNF-α.

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